The goal of this application is to characterize the role that the matrix metalloproteinase enamelysin (MMP-20) plays during dental enamel and tooth development. The enamel proteins (amelogenin, ameloblastin, enamelin) are cleaved soon after they are secreted into the enamel matrix. Enamelysin is expressed simultaneously with the enamel proteins and recombinant enamelysin will cleave recombinant amelogenin at virtually all of the precise sites previously observed in vivo. Thus, enamelysin is an important amelogenin processing enzyme. As for all MMPs, enamelysin contains a propeptide that must be cleaved if the enzyme is to become active. Enamelysin also has a hemopexin domain that is not functionally well defined. An enamelysin knockout (-/-) mouse was engineered to characterize the contribution of enamelysin to tooth and enamel development (Aim 1). The enamelysin -/-mouse is the only MMP -/- mouse with a profound phenotype that survives to breed. Thus, we can define the limits of the enamelysin promoter (Aim 2) so that it may be used to express an enamelysin transgene in the -/- background and revert the -/- phenotype back to normal. Furthermore, because the -/- mouse has a profound phenotype and can breed, we are in possession of the only MMP -/- mouse that can be utilized to characterize the mechanistic function of the MMP propeptide and/or hemopexin domain. So, we propose to introduce two enamelysin promoter transgenes into the -/-background. The first transgene will allow enamelysin to be secreted as an active enzyme (propeptide removed intracellularly) so that we may characterize the role of the enamelysin propeptide in tooth development (Aim 3). The second transgene will encode enamelysin without its' hemopexin domain so that the contribution of the hemopexin domain in tooth development may be characterized (Aim 4). The long-term goals of this project are to contribute to the understanding of enamel formation so that eventually synthetic enamel can be engineered for the repair of damaged (dental caries) or diseased (amelogenesis imperfecta) dental enamel. This application builds on results from a highly productive R29 grant (DE12098).